Journal: Nucleic acids research

Article Title: CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells.

doi: 10.1093/nar/gkac159

Figure Lengend Snippet: Figure 3. A positive correlation was observed between target mRNA level and RxCas13d off-target effects. (A) Drosophila DL1 cells were co-transfected with a constant amount of RxCas13d/guide RNA (50 ng) and mCherry (225 ng) expression plasmids, but variable amounts of eGFP expression plasmid (2, 5, 10, 25 or 50 ng). Empty vector (pUb-3xFLAG MCS (No BsmBI) plasmid) was added as needed so that 500 ng DNA was transfected in all samples. 24 h after transfection, CuSO4 was added and total RNA was isolated after an additional 14 h. RNA expression levels were then analyzed by RT-qPCR (B) or northern blotting (C). (B) RT-qPCR was used to quantify the expression of eGFP mRNA in cells transfected with the RxCas13d plasmid expressing a random guide RNA or a guide RNA complementary to eGFP. For each amount of eGFP plasmid transfected, the relative abundance of eGFP mRNA was normalized to the respective random guide RNA samples. Data are shown as mean ± SD, N = 3. (∗) P < 0.05. (C) Northern blots (20 g of total RNA/lane) were used to quantify the relative expression levels of mCherry and RxCas13d mRNAs. Data are shown as mean ± SD, N = 3. For statistical comparisons, data were compared to the random guide RNA samples. (∗) P < 0.05. n.s., not significant. A complete table of P-values for all comparisons is provided in Supplementary Table S5.

Article Snippet: Drosophila reporter plasmids expressing eGFP (Hy pMT eGFP SV40; Addgene #69911), Laccase2 Exons 1–3 (Hy pMT Laccase2 Exons 1–3; Addgene #91799), and nLuc (Hy pMtnA nLuc SV40; Addgene #132654) under the control of the inducible Metallothionein A promoter were described previously (38–40).

Techniques: Transfection, Expressing, Plasmid Preparation, Isolation, RNA Expression, Quantitative RT-PCR, Northern Blot